Protein drugs are generally formulated for parenteral administration, i.e., injection or infusion, because of their extremely poor bioavailability. Parenteral administration of protein drugs usually requires a visit to the doctor or, in some cases, a hospital. As a result, medical care for patients who require parenteral administration of protein drugs is often expensive and time-consuming. Furthermore, patient compliance is often problematic, especially for those patients who require long-term treatment.
To address this problem, needleless injection technologies, e.g., needleless sub-cutaneous administration, and alternative drug dosage and delivery methods and forms, e.g., dry powder inhalation, skin electroporation, and sustained or controlled release drugs, have been employed.
For use in needleless injection technologies and alternative drug dosage and delivery methods and forms, protein drugs must be manufactured as solid particles to achieve the necessary stability. For many applications, the protein particles to be used must have a well-defined narrow size and morphology. For example, for delivery of a protein drug via inhalation, the diameter of the protein particles to be inhaled must be approximately 2-3 microns, if the main site of action, the alveoli, is to be reached. A number of methods have been employed to prepare micron-sized protein particles, including spray-drying, lyophilization and jet milling. These methods are problematic because they typically denature proteins by heat and mechanical stress. Therefore, there is a need for alternative methods of preparing micron-sized or nanometer-sized protein particles without losing biological activity of the protein. The SPPs, spherical nanocrystalline composite particles and crystalline SPPs according to this invention solve this problem, as well as the problems discussed below.
Ruth et al., Acta Crystallographica D56:524-28 (2000) (“Ruth”), which refers to α-L-iduronidase semi-crystalline spherulites that were made using the hanging drop method of crystallization. The α-L-iduronidase spherulites formed when crystallization solutions at pH 3.0-8.5 were used in the presence of calcium or zinc salts. However, during the process of forming spherulites, the α-L-iduronidase protein underwent a conformational change, possibly due to partial denaturation or unfolding of the α-L-iduronidase protein. The methods of the present invention avoid any change of conformation or resulting loss of biological activity.
U.S. Pat. No. 6,063,910 (the '910 patent), refers to a method of preparing protein microparticles by supercritical fluid precipitation. That method has a number of shortcomings that are overcome by the present invention. The method disclosed in the '910 patent requires suspending the protein of interest in 90% organic solvent, which is not suitable for a number of proteins. Furthermore, the method disclosed in the '910 patent yields particles that are precipitates, unlike the methods of the present invention, which, in some embodiments, could yield SPPs that are crystalline in nature. In addition, the particles resulting from the method of the '910 patent have a diameter of less than 5 microns, while the SPPs, spherical nanocrystalline composite particles or crystalline SPPs of the present invention form particles that range in diameter from about 0.04 to about 200 microns and possibly even larger.
Additional methods of preparing protein particles have also been disclosed, e.g., Bustami R. T. et al., Pharmaceutical Research 17:1360-66 (2000) (“Bustami”). Bustami refers to a method of forming spherical microparticles of proteins using high pressure modified carbon dioxide. The particles formed using the Bustami method are only about 0.1-0.5 microns in diameter, while the method of producing SPPs, spherical nanocrystalline composite particles or crystalline SPPs of the present invention leads to particles that range in diameter from about 0.04 to about 300 microns. Furthermore, in Bustami, the proteins that were formed into microparticles lost up to 60% (for recombinant human deoxyribonuclease (rhDNase)) of their biological activity as a result. Using the methods of the present invention, no loss of biological activity is expected. Finally, the method of Bustami apparently induces protein aggregation, while the method of the present invention does not.
In principle, dried SPPs, spherical nanocrystalline composite particles or crystalline SPPs may also be prepared by lyophilization. See, e.g. Morita T. et al., Pharmaceutical Research 17:1367-73 (2000) (“Morita”). Morita refers to a method of forming spherical fine protein microparticles through lyophilization of a protein-polyethylene glycol (PEG) aqueous mixture. The method in Morita relies on phase separation (unlike the methods herein) followed by lyophilization to yield spheres that have a diameter of 2-3 microns. The methods of the present invention invention lead to the formation of distinct particles that can be isolated by centrifugation, filtration or lyophilization. Also, the SPPs, spherical nanocrystalline composite particles or crystalline SPPs of the present invention form particles that range in diameter from about 0.04 to about 300 microns. Furthermore, the method disclosed in Morita requires the addition of organic solvents, e.g., methylene chloride, to remove the PEG used in a previous step, which, as stated above, is not suitable for a number of proteins. In addition, the SPPs, spherical nanocrystalline composite particles or crystalline SPPs of the present invention are suitable for transfer out of the mother liquor used in their formation and into other solvents, e.g. aqueous isopropyl alcohol. Also, the method disclosed in Morita requires the use of PEG, which may or may not stabilize the protein being used. In contrast, the methods of this invention allow for the use of reagents other than PEG, that may be more capable of stabilizing the protein of interest. For example, the methods of this invention may utilize ammonium sulfate, which generally stabilizes proteins, and which cannot be used in the method disclosed in Morita.
Another limitation of the Morita method is that the disclosed technique involves rapid cooling of the material and can be applied only to freeze stable products. The aqueous solution is first frozen to between −40 and −50° C. Then, the ice is removed under vacuum. Ice formation is usually destructive to the protein crystal lattice, which destabilizes the protein molecule, and sometimes leads to the formation of amorphous precipitate. The methods of the present invention avoid this problem.